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Helicase Loader Complex Reveals Insights Into the Mechanism of Bacterial Primosome Assembly

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ARTICLE Received 20 Jun 2013 | Accepted 22 Aug 2013 | Published 19 Sep 2013 DOI: 10.1038/ncomms3495 OPEN Structure of a helicase–helicase loader complex reveals insights into the mechanism of bacterial primosome assembly Bin Liu1,2, William K. Eliason1,2 & Thomas A. Steitz1,2,3 During the assembly of the bacterial loader-dependent primosome, helicase loader proteins bind to the hexameric helicase ring, deliver it onto the oriC DNA and then dissociate from the complex. Here, to provide a better understanding of this key process, we report the crystal structure of the B570-kDa prepriming complex between the Bacillus subtilis loader protein and the Bacillus stearothermophilus helicase, as well as the helicase-binding domain of primase with a molar ratio of 6:6:3 at 7.5 Å resolution. The overall architecture of the complex exhibits a three-layered ring conformation. Moreover, the structure combined with the proposed model suggests that the shift from the ‘open-ring’ to the ‘open-spiral’ and then the ‘closed- spiral’ state of the helicase ring due to the binding of single-stranded DNA may be the cause of the loader release. 1 Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520, USA. 2 Howard Hughes Medical Institute, New Haven, Connecticut 06510, USA. 3 Department of Chemistry, Yale University, New Haven, Connecticut 06520, USA. Correspondence and requests for materials should be addressed to T.A.S. (email: [email protected]). NATURE COMMUNICATIONS | 4:2495 | DOI: 10.1038/ncomms3495 | www.nature.com/naturecommunications 1 & 2013 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3495 he replication of the bacterial chromosome is initiated at exhibit different quaternary structures4,5. The more recent cryo- oriC where the initiator protein DnaA binds to start the EM structure shows a spiral arrangement of the two interacting Tassembly of the enzymatic replisome machine1. The early hexamers with a split ring, consistent with the possibility of a stages of this process involve the assembly of the primosome and ring-breaking role of the loader5. the formation of a functional primosome2,3. Subsequent to the The translocation of the helicase ring in a 50–30 direction on the remodelling of the replication origin induced by DnaA, the oriC transports primase to the initiation sites and enables the assembly of the bacterial loader-dependent primosome occurs in synthesis of RNA primers. Previous studies using gel filtration, discrete steps and involves at least four different proteins (DnaA, fluorescence, cross-linking and atomic force microscopy indicated helicase, helicase loader and primase) that act in a coordinated that primase DnaG binds to helicase to form a functional and sequential manner. primosome with variable stoichiometries that vary from one to In the Escherichia coli system, the helicase loader protein, three primase molecules to one hexameric helicase ring19,31,32. DnaC, complexed with ATP, binds to hexameric helicase DnaB However, our recent structure of BstDnaB in complex with GDP and forms a DnaB6–DnaC6 complex, which has been confirmed and ssDNA substrate suggests that only one primase is included by cryo-electron microscope (cryo-EM) studies4,5. The loader in the complex when it functions in making RNA primers24. The protein delivers the helicase onto the melted DNA single strands DnaG primase consists of three domains: an N-terminal zinc- of the DnaA–oriC nucleoprotein complex at the origin of binding domain, an RNA polymerase domain and a C-terminal replication2,6. In vivo, this delivery is associated with the helicase-binding domain (HBD)33. Functional interactions initiator protein DnaA2,7, whose amino-terminal domain between DnaB helicase and DnaG primase show that DnaG (NTD) is thought to have a role in loading the helicase and stimulates the ATPase and the helicase activities of DnaB19, and helicase loader complex onto the oriC by interacting with helicase DnaB modulates and increases the synthesis of RNA primers34.It DnaB8. After the loader protein dissociates from the helicase ring, has been believed that loader protein would be ejected from the the NTD of the helicase interacts with the carboxy-terminal helicase and loader complex mounted on the oriC before primase domain (CTD) of the primase and forms a functional primosome binds to the NTD of helicase. However, recent studies that show that synthesizes RNA primers9. Primosome assembly in Gram- the existence of a stable complex between helicase, loader protein positive bacteria is different in the details, including that the and primase15, the synthesis of RNA primers in the presence of corresponding helicase is named DnaC in some bacteria such as the loader protein in vitro14,31 and the effects of ribonucleotides Bacillus subtilis, and the loader protein is DnaI; the assembly of and primase on the release of the loader protein from the helicase the helicase and loader protein complex onto the replication and loader complex35 have suggested that the release of loader origin is assisted by a pair of co-loader proteins DnaB and DnaD protein is coupled with the movement of the helicase ring and the in B. subtilis10–13. synthesis of the RNA primer, and not the initial recruitment of The hexameric helicase DnaC in B. subtilis is inactive primase. in the unwinding activity14 and less stable than the E. coli Although biochemical studies have shed light on the DnaB, and can easily form a mixture of different oligomeric mechanisms of primosome assembly, crystal structures of the states12. However, the stable hexameric DnaB helicase in complexes that occur during primosome assembly are quite B. stearothermophilus (BstDnaB) was observed to form a quite limited. Here we report a crystal structure of BstDnaB helicase stable complex with the loader protein DnaI in B. subtilis in complex with BsuDnaI loader protein and the HBD of (BsuDnaI)15,16. The replicative hexameric DnaB helicase is BstDnaG primase at 7.5 Å resolution. This structure shows how composed of two domains that are connected by one flexible the DnaB helicase interacts with the DnaI loader with a linker17,18. The larger CTD exhibits a classical RecA fold, binds stoichiometry of one hexameric helicase ring binding to three the nucleoside triphosphate and translocates the protein on the loader protein dimers and three HBDs of primase DnaG. DNA, whereas the NTD surrounds the single-stranded DNA Moreover, the structure and the proposed model suggest that the (ssDNA) during DNA unwinding and binds the primase19,20. release of helicase loaders may result from the binding of ssDNA DnaB helicase can bind to ssDNA but not to the oriC in the to the helicase ring, which is accompanied by the shift from the absence of loader protein3,21. The crystal structures of various ‘open-ring’ to the ‘open-spiral’, and then the ‘closed-spiral’ state hexameric helicases in complex with ssDNA or single-stranded ofthehelicasering. RNA show a spiral staircase of ssDNA or single-stranded RNA passing through the central channel of the helicase ring22–24. The DnaI helicase loader belongs to the AAA þ family of Results ATPases25,26. The CTD consists of an AAA þ fold that is Structure determination. The complex of BstDnaB helicase, required for nucleotide and ssDNA binding. Its smaller NTD BsuDnaI loader and the HBD of BstDnaG primase was obtained functions as a molecular switch to regulate the binding of DNA16. by first purifying the DnaB–DnaI complex using gel filtration The NTD of this DnaI loader, which contains a novel zinc- chromatography and then adding the HBD of DnaG (Fig. 1). This binding fold, is not homologous to that of E. coli loader protein procedure is the same as previously reported15. Analysis of the DnaC27, and was found to interact with the CTD of the DnaB proteins in these crystals using mass spectrometry and tryptic helicase in the complex of DnaB with DnaI15. The loading of the digestion confirmed the presence of these three different proteins. hexameric helicase ring onto ssDNA can be achieved by the In addition, another crystal form was also obtained under helicase loader or the primase-like domain of the helicase28. Two different conditions and was shown to contain one hexameric different mechanisms of the loader-dependent loading have been DnaB and three HBDs of DnaG by using molecular replacement. proposed, based on biochemical studies: helicase ring-breaking Further, calculation of the Matthews coefficient confirmed the and ring-making12,29,30. In some species, such as E. coli, helicase existence of hexameric DnaB, hexameric DnaI and three HBDs of loader is believed to aid the parting of a subunit interface in the DnaG in an asymmetric unit. stable helicase ring and thereby break the ring. However, in other The structure was determined by molecular replacement. species, such as B. subtilis, the oligomeric helicases can be Initially, subunits or single proteins were used as search models. assembled into a hexamer ring around DNA by the helicase We then built a new model containing as many components as loader, which acts as a ring maker. There are two reported cryo- possible that were positioned using the cryo-EM density map of EM structures of the E. coli helicase–helicase loader complex that the E. coli helicase and helicase loader complex4 (EMD-1017, 2 NATURE COMMUNICATIONS | 4:2495 | DOI: 10.1038/ncomms3495 | www.nature.com/naturecommunications & 2013 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3495 ARTICLE a Peak 2 160 Peak 2 Peak 1 140 Markers (kDa) 250 150 100 120 75 CTD of DnaB 50 37 100 25 20 Peak 1 15 (mAU) 80 10 280 60 OD BstDnaB CTD of DnaI 40 BsuDnal BstDnaB.BsuDnal 20 0 20 40 60 80 100 120 90° 90° Elution volume (ml) b kers Peak Peak2 Mar1 (kDa) 250 150 60 100 Peak 1 75 50 37 25 40 20 (mAU) 15 10 280 OD 20 BstDnaB.BsuDnal BstDnaB.BsuDnal.HBD Peak 2 0 Figure 2 | Fitting of the CTDs of both BstDnaB and BsuDnaI into the 50 100 150 200 250 300 350 cryo-EM map of the E.
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  • Crystal Structure of Prib, a Component of the Escherichia Coli Replication Restart Primosome

    Crystal Structure of Prib, a Component of the Escherichia Coli Replication Restart Primosome

    Structure, Vol. 12, 1967–1975, November, 2004, 2004 Elsevier Ltd. All rights reserved. DOI 10.1016/j.str.2004.09.004 Crystal Structure of PriB, a Component of the Escherichia coli Replication Restart Primosome Matthew Lopper,1 James M. Holton,2 in vitro conversion of bacteriophage φX174 single- and James L. Keck1,* stranded DNA (ssDNA) to its duplex replicative form 1Department of Biomolecular Chemistry (Schekman et al., 1975). Historically, this system pro- University of Wisconsin Medical School vided a model for lagging strand DNA synthesis wherein 550 Medical Sciences Center the role of the primosome is to repeatedly synthesize 1300 University Avenue short RNA oligonucleotides along the DNA template to Madison, Wisconsin 53706 prime Okazaki fragment synthesis. The seven proteins 2 Physical Biosciences Division comprising the primosome, PriA, PriB, PriC, DnaT, Lawrence Berkeley National Laboratory DnaB, DnaC, and DnaG, were isolated biochemically University of California, Berkeley based on this system (Schekman et al., 1975; Wickner Berkeley, California 94720 and Hurwitz, 1974). Subsequently, it was shown that E. coli possesses two primosomes: one that catalyzes replisome assembly at the bacterial origin of replication Summary (oriC) and involves three of the defined primosome pro- teins (DnaB, DnaC, and DnaG), and a second that initi- Maintenance of genome stability following DNA dam- ates replication at the φX174 origin (primosome assem- age requires origin-independent reinitiation of DNA bly site, pas) and involves all seven proteins. While DnaB replication at repaired replication forks. In E. coli, PriA, (the replicative helicase), DnaC (the helicase loading PriB, PriC, and DnaT play critical roles in recognizing protein), and DnaG (primase) were established as essen- repaired replication forks and reloading the replisome tial factors in chromosomal DNA replication initiating at onto the template to reinitiate DNA replication.